Integral membrane enzymes comprise an important class of biocatalysts which play key roles in a number of processes such as signal transduction, oligosaccharide and lipid biosynthesis, membrane transport, energy coupling, and protein biogenesis. A molecular-level understanding of the mechanisms and structures of these catalysts is desirable if such enzymes are to be controlled and exploited for the public good. The proposed research plan represents the first phase of a long-range project designed to promote a general understanding of these enzymes by examining the detailed mechanism and structure of a small (13.2 kDa) membrane- embedded enzyme, E. coli diacylglycerol kinase (DAGK). The long range goal of this project is to address the following questions: 1. How is the mechanism of DAGK different from that of the better- characterized soluble kinases? What do differences reflect in terms of the constraints placed upon the evolution of DAGK by its interfacial environment? Because much is known about how soluble enzymes execute catalysis, it is important to understand the extent to which this knowledge can be extended to membrane enzymes. 2. What are the thermodynamics of the DAGK reaction pathway and how do these related to the catalytic efficiency of an integral membrane enzyme? In addition, can the overall equilibrium constant for its reaction be substantially perturbed by a change in membrane composition? 3. How do the soluble (adenosine triphosphate) and lipophilic (diacylglycerol) substrates for the forward reaction of DAGK physically reach the active site of the enzyme? This question is of crucial relevance if rational strategies for inhibiting similar enzymes are to be developed. 4. What is the relationship of DAGK's three dimensional structure and membrane topology to the above questions? These four issues shall be probed, when possible, by direct physical observation of the relevant phenomena. Special reliance shall be made upon the use of anisotropic ("solid-state") NMR, which is well-suited for the examination of the structures, dynamics, and interactions of molecules associated with membranes and can be utilized in studies of both model and in vivo systems.